One of the major impediments to effective immunotherapy for cancer has been the influence of negative mmunoregulatory mechanisms on otherwise potent immune responses, including CD4+ CD25+ negativeregulatory T cells (Tregs) contained in the tumor-infiltrating lymphocyte population. Investigators in Project 2 (R. Wang, Leader) recently shifted their research to this problem after finding that some of their newly discovered EBV-EBNA1-derived T-cell epitopes could stimulate both CD4+ effector and Treg cells, the latter having the capacity to suppress na'i've T-cell proliferation and immune responses. Further work to exploit this observation showed that TLR8 ligands (Poly-G oligonucleotides) can directly reverse the suppressive function of Treg cells. Consequently, Wang and his group have postulated that EBV-positive Hodgkin disease (HD) and nasopharyngeal carcinoma (NPC), tumors of wide interest to this program project, are enriched with Treg cells whose suppressive function could inhibit the therapeutic effect of T-cell immunotherapy. They will pursue this concept in the current proposal, using both in vitro and in vivo models. In Aim 1 they intend to establish the subsets and prevalence of Treg cells in clinical tumor samples and blood from patients with HD or NPC and then elucidate their suppressive function and mechanisms in human tumor mouse models. To link TLR8 signaling to Treg suppressive function, they propose in Aim 2 to exploit RNA interference (RNAi)-mediated knockdown and TLR8 transgenic mice to determine the signaling pathways or, at least, the key molecules controlling Treg cell function. Ultimately, in Aim 3, they will test three novel strategies in transgenic mice that may improve antitumor responses to cancer vaccines and CTL-based immunotherapy for EBV-positive tumors: 1) use of TLR8 ligands to block the suppressive function of Treg cells; 2) as in 1 except the ligands will be covalently linked to MHC class II- and l-restricted peptides/proteins to enhance CD4+ and CD8+ T-cell responses; and 3) a combination of 1 and 2. The results of this research will be of considerable interest to investigators in Projects 1, 3 and 4, as the availability of a method to suppress Treg cell function in the tumor microenvironment would nicely complement efforts to render EBV-positive or negative tumor cells resistant to the direct inhibitory effects of cytokines and other factors that can impede the antitumor activity of CTLs. Lay summary - Among the many obstacles to effective immunotherapy for cancer, the negative activity of T cells with the ability to suppress immune responses has been the most difficult to overcome. This group of investigators has identified an agent that can eliminate this barrier in the laboratory and now propose to find the best way to use it to neutralize unwanted T cells in the body.